CN103937782A - Fast extraction method of genomic DNA of single rotifer - Google Patents
Fast extraction method of genomic DNA of single rotifer Download PDFInfo
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- CN103937782A CN103937782A CN201410177155.2A CN201410177155A CN103937782A CN 103937782 A CN103937782 A CN 103937782A CN 201410177155 A CN201410177155 A CN 201410177155A CN 103937782 A CN103937782 A CN 103937782A
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- rotifer
- genomic dna
- lysate
- microliter
- buffer liquid
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Abstract
The invention relates to a fast extraction method of a genomic DNA of a single rotifer. The method comprises the following steps: using two reagents, namely lysates consisting of 25mM NaOH and 0.2mM EDTA (Ethylene Diamine Tetraacetic Acid) and a buffer liquid, namely 40mMTris-HCl at pH 5.0; the fast extraction method comprises the following operating steps: cleaning and starving a rotifer obtained from a natural water body for 72 hours; separating the single rotifer under an anatomical lens; sub-packaging in a 0.2ml EP (electro polished) pipe with the volume of 1 microliter; adding 20 microliter of the lysate to react at 95 DEG C for 30 minute and at 0 DEG C for 5 minutes; then, adding 20 microliter of the buffer liquid and uniformly mixing; and centrifugalizing for 2 minutes at 10000g, and taking the supernatant to obtain a template DNA for PCR (Polymerase Chain Reaction). The method provided by the invention has the advantages that the method is short in time and the accomplishing time is 1 hour; the pollution probability is greatly reduced; the method is precise to analyze. The genomic DNA of the single rotifer can be directly obtained, and the PCR result can be used for sequencing analysis. The low-toxicity basic lysate and buffer liquid are used, so that the damage to the operator is reduced.
Description
Technical field
The invention belongs to the extracting method of genomic dna, particularly a kind of list is only taken turns the rapid extracting method of molitor genomic dna.
Background technology
Wheel animalcule is a kind of small zooplankton being extensively distributed in natural water, and because polypide is small, DNA content is few, thus at present widely method be that single wheel animalcule spread cultivation after several days, after parthenogenesis Population is abundant, then batch extracting DNA analyzes.There is three aspects: deficiency: one, the time is long.Due to the wheel animalcule that will spread cultivation, could obtain the DNA that abundant genetic information is consistent and be used for analyzing, so obtain DNA at least one week consuming time from sampling; Two, contaminated having a big risk.The complex operation that spreads cultivation, middle-chain is subject to ciliate, microbial contamination, directly causes DNA can not be used for analyzing; Three, operate dangerous property.The reagent of traditional phenol-chloroform method mostly has strong corrodibility and toxicity, and operator are formed to potential harm.
Summary of the invention
Technical problem to be solved by this invention is: provide a kind of list only to take turns the rapid extracting method of molitor genomic dna.Reach short in the time, Pollution risk is low, isolates wheel animalcule to DNA under low murder by poisoning condition.
Technical scheme of the present invention is:
List is only taken turns a rapid extracting method for molitor genomic dna, it is characterized in that: use lysate: 25mMNaOH, 0.2mMEDTA and damping fluid: 40mMTris-HCL, two kinds of reagent of pH5.0, operating process is: the wheel animalcule obtaining in natural water, through cleaning, after hungry 72 hours, separating list only individual under anatomical lens, is distributed in 0.2mlEP pipe, volume 1uL, add 20uL lysate, 95 DEG C of 30min, 0 DEG C of 5min, add again 20uL damping fluid, mix; The centrifugal 2min of 10000g, gets supernatant, can obtain the template DNA for PCR.
Effect of the present invention and advantage:
Present method can be extracted the genomic dna of single wheel animalcule, and its advantage is:
(1) time is short.Be about 1 hour from separation wheel animalcule to the DNA extraction deadline;
(2) Pollution risk is low.From being separated to, to obtain DNA step few, and the time period, contaminated probability reduces greatly;
(3) precisely analyze.The genomic dna that can directly obtain single wheel animalcule, PCR result can be used for sequencing analysis;
(4) low murder by poisoning.Use basic lysate and the damping fluid of low toxicity, auxiliary by temperature, reduce the harm to operator.
Embodiment
List is only taken turns a rapid extracting method for molitor genomic dna, and this law is the hotshot method after improvement, needs two kinds of reagent of lysate (25mMNaOH, 0.2mMEDTA) and damping fluid (40mMTris-HCL, pH5.0).Operating process is: the wheel animalcule obtaining in natural water, through cleaning, after hungry 72 hours, separating list only individual under anatomical lens, is distributed in 0.2mlEP pipe, and volume 1uL, adds 20uL lysate, 95 DEG C of 30min, and 0 DEG C of 5min, then add 20uL damping fluid, mix.The centrifugal 2min of 10000g, gets supernatant, is the template DNA that can be used for PCR.
Present patent application is by special subsidize " the complete gordian technique joint study of Potable Water Conservation restoration of the ecosystem " (item number: 2013DFA71340) of national International Sci & Tech Cooperation
InternationalScience&TechnologyCooperationProgramofChina(GrantNo.2013DFA71340)。
Claims (1)
1. list is only taken turns a rapid extracting method for molitor genomic dna, it is characterized in that: use lysate: 25mMNaOH, 0.2mMEDTA and damping fluid: 40mMTris-HCL, two kinds of reagent of pH5.0, operating process is: the wheel animalcule obtaining in natural water, through cleaning, after hungry 72 hours, separating list only individual under anatomical lens, is distributed in 0.2mlEP pipe, volume 1uL, add 20uL lysate, 95 DEG C of 30min, 0 DEG C of 5min, add again 20uL damping fluid, mix; The centrifugal 2min of 10000g, gets supernatant, can obtain the template DNA for PCR.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410177155.2A CN103937782A (en) | 2014-04-29 | 2014-04-29 | Fast extraction method of genomic DNA of single rotifer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410177155.2A CN103937782A (en) | 2014-04-29 | 2014-04-29 | Fast extraction method of genomic DNA of single rotifer |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103937782A true CN103937782A (en) | 2014-07-23 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410177155.2A Pending CN103937782A (en) | 2014-04-29 | 2014-04-29 | Fast extraction method of genomic DNA of single rotifer |
Country Status (1)
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| CN (1) | CN103937782A (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101696408A (en) * | 2009-10-23 | 2010-04-21 | 厦门大学 | Method for extracting DNA from single eggs of copepods |
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2014
- 2014-04-29 CN CN201410177155.2A patent/CN103937782A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101696408A (en) * | 2009-10-23 | 2010-04-21 | 厦门大学 | Method for extracting DNA from single eggs of copepods |
Non-Patent Citations (5)
| Title |
|---|
| GABALDON ET AL.: "Morphological Similarity and Ecological Overlap in Two Rotifer Species", 《PLOS ONE》 * |
| MONTERO-PAU ET AL.: "Application of an inexpensive and high-throughput genomic DNA extraction method for the molecular ecology of zooplanktonic diapausing eggs", 《LIMNOL.OCEANOGR：METHODS》 * |
| TRUETT ET AL.: "Preparation of PCR-Quality Mouse Genomic DNA with Hot Sodium Hydroxide and Tris(HotSHOT)", 《BIOTECHNIQUES》 * |
| 徐在宽等: "《泥鳅 黄鳝无公害养殖 重点、难点与实例》", 31 October 2005, 科学技术文献出版社 * |
| 王和全等: "轮虫休眠卵的采集、加工和储存技术", 《河北渔业》 * |
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| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
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| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20140723 |
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| RJ01 | Rejection of invention patent application after publication |